Applied molecular biology Book: Glick, Pasternak, Molecular Biotechnology, Principles and application of recombinant DNA Biotechnology Molecular genetics

What Is Biotechnology? Using scientific methods with organisms to produce new products or new forms of organisms Any technique that uses living organisms or substances from those organisms to make or modify a product, to improve plants or animals, or to develop microorganisms for specific uses

Beer is an ancient foodstuff Ancient beer was not just drink, but food Thick drink with high caloric value as well as alcohol

Yeast cells Both beer and bread were developed around the same time in the middle east Early bread was flat, but when wild yeast contaminated the dough, a fluffier, sweeter bread was created Beer arose out of the liquid soaked bread

Cheese & yogurt also came about due to microbial contamination

Classical Biotechnology Refinement of fermentation techniques during 18 th and 19 th C. During 20 th C. fermentation expanded to the production of: Glycerol Acetone Butanol Lactic acid Citric acid

Herbal plants have been used since ancient times Even today, 25% of our common medicines contain at least some compounds obtained from plants Why do plants create these compounds? Protection from herbivory and predation Allelopathy - plants secrete toxins from their roots that prevent the germination of other plants in their root zone Biopharmaceuticals

Alkaloids: Over 5,000 alkaloids have been identified in numerous plant families, most in the angiosperms Contain nitrogen Alkaline Bitter Physiological effect on animals, often on nervous system Names of most alkaloids end in "...ine"

Common Medicinal Alkaloids & their Sources: MorphinePoppies CaffeineCoffee/Tea NicotineTabacco EmetineIpecac AtropineBelladonna QuinineCinchona Tree

During 19 th C. quinine was critical to British colonial expansion Extracted from the bark of the cinchona plant Not enough could be extracted, another source was needed

Bayer discovered way to synthesize acetylsalicylic acid Known under its trade name, Aspirin

Penicillin

Penicillium mold In 1928 Alexander Fleming noticed something odd about a petri dish contaminated with mold The mold seemed to kill the bacteria Fleming was unable to isolate the bactericidal action

In 1940 Norman Heatley finally showed that penicillin could stop infection Mice were infected with streptococcus bacteria Half were given penicillin. Those receiving penicillin survived, those that didnt died. No penicillin penicillin 24 hrs. later No penicillin penicillin

1 st human patient was a policeman with staphylococcal & streptococcal infections which had already taken part of his face and an eye He began to recover, but later died 2 nd patient was a 15 yr. old boy septic from a hip operation Two days after receiving penicillin his temperature dropped back to normal after being at 100° for 2 wks

By D-Day the U.S. was making millions of doses. Unfortunately, neither Heatley nor his boss patented the discovery. This was done by U.S. firms. Thus for 25 yrs. England had to pay royalties on its own discovery.

Genetics - historical perspective Practical genetics 7,000 yeas ago corn breeding - Central America rice breeding - China horse pedigree - Babylon Genetics - science - Mendel

A domesticated animal is one which has been bred in captivity Thru artifical selection they are modified from their ancestors for use by humans BeforeAfter

Wolf/Dog domestication lead to: Alteration in body size Reduction in skull & tooth size Shortening of the jaw bones Affection for humans Variation in coat color Tendency towards barking By 6000BC dog skeletons are found along side human remains

Modern sheep have been bred not to lose their wool

Most domesticated species arose in SW Asia or China Of the ~150 species of terrestrial non-carnivores >100 lbs, only 14 have been domesticated 13 are of Eurasian origin, one from mesoamerica None derive from Australia or sub-Saharan Africa

Desirable Characteristics for Domestication of an Animal Species Value to humans as food, draft, fiber, or hunting Large herbivores offer energy use advantages Rapidly reach their desired size Must be able to breed in captivity Good disposition & social structure

The switch from hunter-gatherer to farmer took place between & years ago Both Eurasia & the Americas developed large numbers of domesticated crops The development of agriculture required changes in wild plants such that they were amendable to cultivation Many of these changes were either brought about by humans or were capitalized by them Plant Domestication

Example Wheat is a grass spread seeds called grains Mutants developed that did not lose seed This made it easier for humans to collect

Hybridization played a role in the evolution of modern grains

Changes in corn size from 5000 BCE to 1500CE The evolution of modern corn took several thousand years Selection for larger ears by mesoamericans created modern corn by the time Europeans had reached the Americas

Mutation responsible for this change has been identified It is not a change in a gene itself, rather it is a decrease in the expression of the gene tb1

Later Concepts Not until 34 years after its publication did Mendels work receive additional attention, with publications in 1900 by three Botanists: Hugo de Vries, Carl Correns, and Erich von Tsernak; Walter Sutton first integrated the concepts of chromosomes with Mendels laws, in studies of grasshopper reproduction and cell division and concluded that Mendels heritable factors must be on the chromosomes – T.H. Morgan began his work with fruit flies, ultimately mapping gene locations.

First Structure By 1910 actual components known (nucleotides) –Phoebus Levene proposed a tetranucleotide structure for DNA Tetranucleotide repeat of ATCG Own data showed nucleotides not in 1:1:1:1 ratio Differences probably experimental error…

So… If DNA was a single covalently bonded tetranucleotide structure then it couldnt easily encode information Proteins, on the other hand, had 20 different amino acids and could have lots of variation Most geneticists focused on transmission genetics and passively accepted proteins as being the likely genetic material

T. H. Morgans Fruit Flies s

Frederick Griffith, 1928 Transformation of Bacteria Transforming factor ?

Avery, McCarty and MacLeod After 10 yrs of effort published work using Griffiths approach to assay for the genetic material –Used Cell-free extract of S cells From 75 liters of cell culture obtained mg of active factor Proteases, RNases, DNases, etc. Transforming factor is DNA

Erwin Chargaff Digested many DNAs and subjected products to chromatographic separation Results –A = T, C = G –A + G = C + T (purine = pyrimidine) –A + T does not equal C + G Members of a species similar but different species vary in AT/CG ratio

X-ray Crystallography of DNA Franklin and Wilkins

Watson and Crick 1953 propose double helix model –Right-handed double helix Collaborated at Cambridge, England.

Impact Article in Nature –It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copy mechanism for the genetic material Second paper 2 months later describes semiconservative replication and that mutations must change bases in DNA (information encoded in the bases and their order) DNA became the genetic material…

DNA Replication